Abscisic Acid increases terrestrial plant cell resistance to hydrostatic pressure.

Cells of the terrestrial plant species bromegrass (Bromus inermis L.) are not naturally adapted to withstand the hydrostatic pressures encountered in aquatic environments. However, after treatment with the natural plant growth hormone abscisic acid (75 micromolar), bromegrass cells survived a hydrostatic pressure of 101.3 megapascals, approximating the limits of ocean depth (10,860 m). The increased resistance to hydrostatic pressure from 1 to 7 days of abscisic acid treatment paralleled the induced elevation of cell tolerance to freezing stress.

Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells.

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 x 10(-5) molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23 degrees C) for 7 days acclimated to -28 degrees C (LT(50)) when treated with ABA, or to -5 degrees C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H(2)O) or as grams water per gram dry weight (gram H(2)O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H(2)O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H(2)O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H(2)O/10(6) cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10(6) cells increased to 3.3 milligram/10(6) cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10(6) cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur.

Freezing of water in red-osier dogwood stems in relation to cold hardiness.

Studies of stem water in red-osier dogwood (Cornus stolonifera Michx.) using nuclear magnetic resonance spectroscopy indicated that most freezing occurs at temperatures above -30 C in cold-hardy and tender stems. Hardy and tender stems had about the same amount of unfrozen water at -40 C (0.28 gram of water per gram dry weight). When hardy stems were slowly cooled below -20 C, the temperature below which little additional freezing occurs, they survived direct immersion in liquid N(2) (-196 C). Fully hardy samples not slowly precooled to at least -15 C did not survive direct immersion in liquid N(2). The results support the hypothesis that cooling rate is an unimportant factor in tissue survival at and below temperatures where there is little freezable water.

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx.

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from -3 C to -40 or -50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate-requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O(2)-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.

Water Permeability and Cold Hardiness of Cortex Cells in Cornus stolonifera Michx.-A Preliminary Report.

The relationship of freezing resistance to water permeability of cortex cells was studied in stems of red osier dogwood (Cornus stolonifera Michx.). Permeability was estimated by determining the diffusion flux of tritiated water from cortex slices previously equilibrated in tritiated water. Energy of activation and diffusion time comparisons of tritiated water flux from living cortex slices and slices killed by immersion in liquid N(2) verified that intact membranes of uninjured cortex cells limited water flux.Water permeability of living phloem and cortical parenchyma cells increased during the initial (photoperiodically induced) phase of cold acclimation. This accompanied an increase in hardiness from -3 to -12 C. Little if any further increase in permeability was noted during subsequent acclimation to below -65 C.Permeability measurements on nonhardy cortex samples yielded consistent results, but measurements on samples from hardy twigs were often difficult to reproduce. This unexplained variability precludes specific conclusions, but the tritiated water diffusion flux technique may provide an alternative to traditional plasmolytic techniques in studying water permeability in woody plant tissues.

Supercooling in overwintering azalea flower buds.

Differential thermal analysis and nuclear magnetic resonance spectroscopy experiments on whole flower buds and excised floral primordia of azalea (Rhododendron kosterianum, Schneid.) proved that supercooling is the mode of freezing resistance (avoidance) of azalea flower primordia. Increase in the linewidth of nuclear magnetic resonance spectra for water upon thawing supports the view that injury to the primordia occurs at the moment of freezing. Nonliving primordia freeze at the same temperatures as living primordia, indicating that morphological features of primordial tissues are a key factor in freezing avoidance of dormant azalea flower primordia. Differential thermal analyses was used to study the relationship of cooling rate to the freezing points of floral primordia in whole flower buds. At a cooling rate of 8.5 C per hour, primordia in whole buds froze at about the same subfreezing temperatures as did excised primordia cooled at 37 C per hour. At more rapid cooling rates primordia in intact buds froze at higher temperatures.

Effects of Red and Far Red Light on the Initiation of Cold Acclimation in Cornus stolonifera Michx.

Red and far red light distinctly influence the initial phytochrome-mediated phase of cold acclimation in red-osier dogwood (Cornus stolonifera). Under controlled conditions, short days and end-of-day far red light exposure after long days promote growth cessation, cold acclimation, and subsequent cold hardening of dogwood stems in response to low temperature. Nuclear magnetic resonance absorption spectra of the water in internode stem sections imply that the short day-induced phase of cold acclimation involves a change in tissue hydration, at least in part, due to a substantial reduction in bulk phase water as a result of senescence and loss of water from the pith. Seasonal responses to light and an attempt to induce early acclimation under natural conditions with end-of-day far red light are discussed.

Short term increases in the cold tolerance of red osier dogwood stems induced by application of cysteine.

Bark tissues of Cornus stolonifera stems, treated with cysteine at 24 hours after treatment, survived exposure to -11 C (the tissue temperature) with little or no injury. An initiation of increase in the cold tolerance was usually observed when plants were treated with cysteine at 12 hours after treatment. Neither plants at 36 or 48 hours after treatment nor plants 12 hours before treatment had shown increases in the cold tolerance. They were killed below -5 C, which was the survival temperature of untreated control plants. Two weeks or more of short day induction before cysteine application were required for a significant effect of short term 5 C increase in the cold tolerance.

The mechanism of freezing injury in xylem of winter apple twigs.

In acclimated winter twigs of Haralson apple (Pyrus Malus L.), a lag in temperature during cooling at a constant rate was observed at about -41 C by differential thermal analysis. The temperature at which this low temperature exotherm occurred was essentially unaffected by the cooling rate. During thawing there was no lag in temperature (endotherm) near the temperature at which the low temperature exotherm occurred, but upon subsequent refreezing the exotherm reappeared at a somewhat higher temperature when twigs were rewarmed to at least -5 C before refreezing. These observations indicate that a small fraction of water may remain unfrozen to as low as -42 C after freezing of the bulk water in stems. The low temperature exotherm was not present in twigs freeze-dried to a water content below 8.5% (per unit fresh weight), but it reappeared when twigs were rehydrated to 20% water. When freeze-dried twigs were ground to a fine powder prior to rehydration, no exotherm was observed. Previous work has shown that the low temperature exotherm arises from xylem and pith tissues, and that injury to living cells in these tissues invariably occurs only when twigs are cooled below, but not above the temperature of the low temperature exotherm. This study revealed that the low temperature exotherm resulted from the freezing of a water fraction, that the freezing of this water was independent of the freezing of the bulk water, that the exotherm was associated with some gross structural feature but not the viability of the tissue, and that injury to living cells in the xylem and pith was closely and perhaps causally related to the initial freezing of this water.

Freezing injury in potato leaves.

Time-temperature profiles of freezing leaves from frost-resistant (Solanum acaule Bitt.) and frost-susceptible (Solanum tuberosum L. subsp. tuberosum Hawkes) types of potatoes did not reveal any major differences. The pattern of change in resistance of leaves to low voltage, low frequency current during freezing was different in the frost-resistant and susceptible leaves. In tissue sections from both types of leaves, cells freeze extracellularly at cooling velocities lower than 5 C per minute. Cells from leaves of resistant plants showed a higher osmotic pressure but not a higher water permeability than those from susceptible plants. The extent of injury caused by even very slow freezing was greater than that caused by equivalent isopiestic desiccation, particularly in susceptible leaves. The higher osmotic pressure in cells of leaves from resistant plants can account for the greater desiccation resistance but not for the frost resistance observed.

Influence of decenylsuccinic Acid on water permeability of plant cells.

Decenylsuccinic acid altered permeability to water of epidermal cells of bulb scales of Allium cepa and of the leaf midrib of Rhoeo discolor. Water permeability, as determined by deplasmolysis time measurements, was related to the dose of undissociated decenylsuccinic acid (mm undissociated decenylsuccinic acid x minute). No relationship was found between permeability and total dose of decenylsuccinic acid, or dose of dissociated decenylsuccinic acid, suggesting that the undissociated molecule was the active factor in permeability changes and injury.At doses which did not damage cells (0.0008 to 0.6 [mm of the undissociated molecule x minute]) decenylsuccinic acid decreased water permeability. At higher doses (e.g., 4 to 8 [mm x minute]) injury to cells was common and decenylsuccinic acid increased permeability. Doses above the 10 to 20 (mm x minute) range were generally lethal. The plasmolysis form of uninjured cells was altered and protoplasmic swelling occasionally was observed. The dose-dependent reversal of water permeability changes (decreased to increased permeability) may reflect decenylsuccinic acid-induced changes in membrane structure. Reported effects of decenylsuccinic acid on temperature dependence of permeability and frost resistance were not verified.

Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves.

Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.Exposure of hardy boxwood plants to warm temperatures resulted in a rapid loss in cold resistance and a rapid synthesis of nucleic acids as judged by (32)P incorporation.Following a killing frost to Korean boxwood leaves, there was a rapid decrease in all nucleic acid fractions which was attributed to nuclease activity. Within 5 hours there was no measurable soluble RNA and ribosomal RNA. Tenaciously bound RNA was somewhat more persistent.

Relationship of Electrical Conductance at Two Frequencies to Cold Injury and Acclimation in Cornus stolonifera Michx.

The ratio of electrical conductance measured at two frequencies can be used to predict the cold hardiness of stem sections of Cornus stolonifera Michx. during the first stage of cold acclimation. Electrical conductance at 50 hertz divided by electrical conductance at 100 kilohertz gave a better estimate of hardiness than measurements at either frequency alone. The observed increase in the electrical conductance ratio as hardiness increased is consistent with an increase in membrane permeability. After plants were exposed to nonlethal frost, hardiness increased rapidly, and the relation between the conductance ratio and hardiness changed. This change indicates that ice crystallization induces a significant physiological alteration in the plants. Contrary to expectations, stem sections exposed to lethal temperatures could not consistently be separated from sections exposed to nonlethal temperatures by electrical conductance ratio measurements made immediately after thawing.

Induction of Cold Acclimation in Cornus stolonifera Michx.

A warm (20 to 15 Celsius day or night) preconditioning treatment enhanced cold acclimation of Cornus stolonifera bark under short-day conditions when plants were preconditioned for at least 4 weeks. Warm preconditioning inhibited the acclimation of plants subjected to long photoperiods. Removing leaves from plants exposed to low temperatures and short days inhibited acclimation. Removal of buds did not affect acclimation. Plants did not acclimate unless they were exposed to at least 4 weeks of short photoperiods prior to defoliation. Plants began to acclimate to cold at the time of growth cessation but not before. When half of the leaves were removed from plants, the defoliated and foliated branches both acclimated as well as branches on completely foliated plants. Girdling the phloem between foliated and defoliated branches prevented acclimation of the latter regardless of the position of the girdle in relation to the root system and the defoliated branch. When all of the leaves of plants were covered with aluminum foil to exclude light after 0 or 4 weeks of exposure to short days, the results resembled a defoliation study, i.e., plants with leaves covered at the start of the experiment failed to acclimate, and those covered after 4 weeks acclimated to some extent but less than uncovered control plants. Under longday conditions plants with all leaves covered failed to acclimate, and plants with none or half of their leaves covered acclimated equally and to a limited extent. Under short-day conditions, however, the covered branches of partially covered plants acclimated more than their uncovered counterparts or branches of totally uncovered plants.

Cold Resistance and Injury in Woody Plants: Knowledge of hardy plant adaptations to freezing stress may help us to reduce winter damage.

It is interesting that plants can eventually acclimate fully in response to low temperatures in the absence of inductive photoperiods (67). This suggests that there is more than one route to resistance or more than one ignition key to start the machinery. In either case, the ability of plants to acclimate in response to more than one environmental stimulus provides adaptive flexibility which enhances survival potential.

The environmental control of cold acclimation in apple.

The role of photoperiod and temperature in the cold acclimation of living Haralson apple (Pyrus malus L.) bark was studied in the autumn under field conditions in Minnesota. Whole trees, or different parts of the same tree, were exposed to either natural conditions, artifically lengthened days, or artificially warmed nights, or they were subjected to manual leaf removal. The results indicate that acclimation occurs in two stages which are induced by short days and frost (or low temperature), respectively. Leaves were stimulated by short days to produce translocatable substance(s) which promoted cold acclimation of the living bark. Leaves of plants grown under long days were the source of a translocatable substance(s) which inhibited acclimation. The second stage of hardiness, induced by frost (or low temperature), did not involve translocatable factors.Inductive short days could overcome the effect of high temperatures, and low temperatures could overcome the effect of noninductive long days in promoting the first stage of acclimation to -30 C. Frost was necessary for maximum hardiness to -55 C. Plants grown in a greenhouse, in the autumn, under long days and high temperatures acclimated slightly in spite of the noninductive conditions. Short days and frost (or low temperatures) appeared to regulate different and independent endogenous acclimation processes.